Abstract The picornaviruses are a family of small positive sense single stranded RNA viruses that cause a wide range of diseases at an annual cost well into the hundreds of million dollars. Members include acute hepatitis A virus, the heart disease causing coxsackie B3 virus, rhinoviruses that cause more than half the occurrences of the common cold, and the paralyzing poliovirus. These viruses share a common life cycle where their RNA replication and viral assembly occurs in large membrane anchored replication complexes assembled on the surfaces of vesicles derived from the endoplasmic reticulum. The replication process is driven by a virally encoded RNA dependent RNA polymerase, the 3Dpol protein, that is responsible the synthesis of all viral RNA. This research project is focused on the structure and assembly of viral replication centers, where we use poliovirus and coxsackievirus as our main experimental system. We have previously solved the crystal structures of the 3Dpol proteins from both these viruses and elucidated the molecular mechanism behind the proteolytic activation of these proteins upon cleavage from the viral 3CDpro precursor protein. We are continuing our studies of picronaviral replication proteins by focusing on the structural changes associated with the formation of the 3Dpol elongation complex and on understanding how mutations in the polymerase affect viral RNA replication both in vitro and in vivo. One new aspect of the project is focused on the structure of the membrane associated viral 2C protein that is responsible for host cell membrane rearrangements that lead to the formation of the vesicles upon which the replication complexes are formed.